Numerous cell surface receptors transduce signals through heterotrimeric GTP binding proteins (G proteins). The alpha subunit of these proteins is a molecular switch, cycling between GDP-bound (inactive) and GTP-bound (active) forms. The purpose of this study is to characterize the intracellular regulation of G-protein-mediated signal transduction. GTPase activity of the alpha subunit is enhanced by a novel family of regulators of G protein signaling (RGS proteins), resulting in inhibition of Gi and Gq-coupled signaling. This project studies specifically the interaction between RGS proteins and G proteins and the resultant control of G protein function. RGS proteins demonstrate little specificity for Gi and Gq subunits in vitro, yet they aparently discriminate between G-protein-coupled receptors (GPCRs) linked to the same G-alpha in some cells. To address the issue of receptor specificity directly, fusion proteins consisting of different GPCRs fused to various G-alpha subunits were constructed and expressed in mammalian cell lines. Receptor-stimulated GTPase activity of membrane preparations is then studied in the presence of RGS proteins. RGS4 was shown to selectively regulate the GTPase activity generated by the stimulation of alpha-2 adrenoceptor fused to Gi-alpha 2 and G-alpha o but not Gi-alpha 1 or 3.Regulation of RGS activity was also studied. RGS16 underwent tyrosine phosphorylation by the p60 src kinase and by the Epidermal Growth Factor (EGF) receptor. Mutation of conserved tyrosine residues blocked tyrosine phosphorylation of RGS16, and the tyrosine mutant was unable to function as a GTPase activating protein (GAP) for Gi-alpha in vitro or in vivo. RGS16 was shown to interact with the G protein G-alpha 13 and inhibit G13-mediated signaling in a GAP-independent manner. A novel RGS protein with a divergent RGS domain was cloned, D-AKAP2, which also contains a protein kinase A (PKA) anchoring (AKAP) domain. D-AKAP2 was shown to interact with activated forms of G-alpha 12 and 13 and to facilitate their activation of PKA, which is an undescribed signaling pathway.